A conserved biological response to double-stranded RNA mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, encoded by transposable elements and viruses, respectively. This silencing response, known variously as RNA interference (RNAi) or post-transcriptional gene silencing (PTGS), also serves to negatively regulate the expression of cellular protein-coding genes. The goal of the proposed research is to understand the molecular mechanism of RNAi, and identify those aspects of cell and organismal regulation that are controlled by RNAi-related activities. We are addressing these issues by studying RNAi in Drosophila melanogaster. Injection or expression of double-stranded RNA in Drosophila serves as a trigger that causes cells to specifically degrade homologous mRNA transcripts. Our approach is to identify essential components of the RNAi mechanism by isolating and characterizing mutations that cause the RNAi response to be weaker than normal. These studies have thus far led to the identification of eight genetic loci that are candidates to encode proteins acting at various steps in the RNAi process. We have molecularly identified two of these genes. One, aubergine, encodes a member of the PIWI/PAZ gene family that includes several other members implicated in PTGS. The other, spindle-E, encodes a DExH-box protein, which may act as an RNA helicase. Both spindle-E and aubergine are also required for translational control in oocytes. Our studies have further linked RNAi with mRNA translation, in that oocyte mRNA transcripts appear to require active translation in order to be target substrates for the RNAi pathway. The goal of the proposed research is to further decipher the mechanism of RNAi by: 1) completing the mutagenesis screen for genes required for RNAi in Drosophila, 2) characterizing the roles of the RNAi genes in controlling RNAi and other biological processes in vivo, 3) determining the biochemical function of RNAi gene products by in vitro analysis of mutants, and 4) cloning and molecular characterization of RNAi genes. The ability of RNAi to regulate viral, transposon, and organismal gene expression suggests that an understanding of RNAi will reveal basic control mechanisms that influence many aspects of biology. Moreover, RNAi has been used experimentally to probe gene function and holds promise as a therapeutic model, making its basic understanding an important biomedical research goal. [unreadable] [unreadable]